Phytochemical profiling, antiviral activities, molecular docking, and dynamic simulations of selected Ruellia species extracts

The antiviral properties of the flowering aerial extracts of Ruellia tuberosa and Ruellia patula were investigated through phytochemical profiling via LC–MS/MS and HPLC techniques. Qualitative LC–MS/MS analyses identified seventy-seven metabolites from both Ruellia species. R. tuberosa had the highest phenolic content (49.3%), whereas R. patula had the highest flavonoid content (57.8%). Additionally, quantitative HPLC investigations of the compounds identified by LC–MS/MS were performed using the available standard compounds. The main constituents in the R. tuberosa extract was found to be catechin (5321.63 µg/g), gallic acid (2878.71 µg/g), and ellagic acid (2530.79 µg/g), whereas the major compounds in the R. patula extract was found to be rutin (11,074.19 µg/g) and chlorogenic acid (3157.35 µg/g). Furthermore, the antiviral activities of both Ruellia species against HAdV-40, herpes simplex type 2 and H1N1 were evaluated. These findings demonstrated that R. tuberosa was more active than R. patula against all tested viruses, except for the HSV-2 virus, against which R. patula showed greater activity than R. tuberosa, with IC50 values of 20, 65, 22.59, and 13.13 µg/ml for R. tuberosa flowering aerial parts and 32.26, 11.66, and 23.03 µg/ml for R. patula flowering aerial parts, respectively for HAdV-40, herpes simplex type 2, and H1N1. Additionally, computational docking and molecular dynamics simulations were used to assess the molecular interactions between the bioactive compounds and specific viral targets. The combined findings from the in-vitro and in-silico experiments comprehensively evaluated the antiviral activities of both Ruellia species extracts.


LC-ESI-TOF-MS analysis of the aq.-Ethanolic extract
Sample preparation A 1:1:10 mixture of water, methanol, and acetonitrile (H 2 O: MeOH: ACN) was used to prepare a stock solution from 50 mg of the previously obtained aqueous-ethanolic extract from Ruellia species.The sample was completely dissolved using vortexing and 10 min of ultrasonication at 30 kHz.To prepare the injection solution, a small portion (20 µL) of the stock solution was diluted with 1000 µL of H 2 O: MeOH: ACN (2:1:1) and centrifuged at 10,000 rpm for 5 min.Finally, 10 µL (equivalent to 1 µg/mL) of the solution was injected for LC-MS analysis.Blank, quality control, and internal standard (IS) samples were also analyzed alongside the sample for experimental confidence.Notably, both positive and negative ionization modes were used during the LC-MS analyses 28 . Vol.:(0123456789)
The following sequence was used for gradient elution: 0-20 min, 10% B; 21-25 min, 90% B; 25.01-28 min, 10% B; and finally, 90% B for column equilibration.A Triple TOF 5600 + system with a Duo-Spray source running in the ESI mode (AB SCIEX, Concord, ON, Canada) was used for the mass spectrometry (MS) experiments.In the positive mode, the sprayer capillary and declustering potential voltages were 4500 V and 80 V, respectively, and in the negative mode, they were − 4500 V and − 80 V, respectively.Gas 1 and gas 2 were set at 40 psi, the curtain gas was at 25 psi, and the source temperature was fixed at 600 °C.The collision energy was set at 35 and − 35 V for the positive and negative modes, respectively with a 20 V CE spread and an ion tolerance of 10 ppm.Information-dependent acquisition (IDA) protocol was used to run the Triple TOF 5600+.Analyst-TF 1.7.1 was used to build batches for the gathering of MS and MS/MS data.Data from both full-scan MS and MS/MS were simultaneously gathered using the IDA technique.High-resolution survey spectra spanning from 50 to 1100 m/z were employed in the technique, and the mass spectrometer was set up to detect survey scans every 50 ms.Following each scan, the top 15 strong ions were chosen to obtain MS/MS fragmentation spectra 28 .

LC-MS data processing
The material was thoroughly examined using small molecules and non-targeting methods with the open-source MS-DIAL 3.70 program.ReSpect-positive (2737 records) or ReSpect-negative (1573 records) databases were used as reference databases based on the acquisition mode.To gather the data for peak detection, the following search parameters were used: minimum peak height, 100 amplitude; mass slice width, 0.05 Da; smoothing level, 2 scans; minimum peak width, 6 scans; and identification for alignment.The tolerances were as follows: MS1 and MS2, 0.2 Da each; retention time, 0.05 min; and MS1, 0.25 Da.For feature (peak) confirmation from the total ion chromatogram (TIC), the MS-DIAL output was used once more to run on PeakView 2.2 with the MasterView 1.1 package (AB SCIEX).The criteria used were aligned features with a signal-to-noise ratio greater than 5 and sample intensities greater than 5 (blank) 28 .

Antiviral assessment
Assessment of the antiviral activity against human adenovirus type 40 (HAdV-40)  The assessment was conducted using Hep-2 cells and human adenovirus type 40.Hep-2 cells were cultured in DMEM supplemented with 0.1% antibiotic/antimycotic solution and 10% fetal bovine serum.Fetal bovine serum, trypsin-EDTA, DMEM, and antibiotic and antimycotic solutions were obtained from Gibco BRL (Grand Island, NY, USA).Using the recently described cytopathic (CPE) inhibitory effect, antiviral activity and cytotoxicity were assessed using the crystal violet method.To summarize, one day before infection, hep-2 cells were plated at a density of 2 × 104 cells/well in a 96-well culture plate.The following day, the culture media was removed, and phosphate-buffered saline was used to wash the cells.The crystal violet method was utilized to determine the infectivity of human adenovirus type 40.This method allowed for calculating the percentage of cell viability while also monitoring CPE.Mammalian cells were exposed to 0.1 ml of a diluted human adenovirus type 40 viral suspension comprising CCID50 (1.0 × 10 4 ) of the virus stock.Three to four days after infection, this dosage was chosen to result in the expected CPEs.The cells were treated with compounds by adding 0.01 ml of media with the required extract concentration.The antiviral activity of each test sample was assessed using a concentration range of 0.1-1000 μg/ml that had been diluted twice.Both the cell controls (noninfected, nondrug-treated cells) and the viral controls (virus-infected, nontreated cells) were included.The culture plates were cultured in 5% CO 2 at 37 °C for 96 h.The progression of cytopathic effects was observed via light microscopy.After being washed with PBS, the cell monolayers were fixed and stained with a 0.03% crystal violet solution in 2% ethanol and 10% formalin.Following cleaning and drying, the optical density of each well was measured using a spectrophotometer set at 570/630 nm.Pauwels et al. provided the following equation, which was used to determine the percentage of antiviral activities of the test compounds: antiviral activity = [(mean optical density of cell controls − mean optical density of virus controls)/(optical density of test − mean optical density of virus controls)] × 100%.The 50% CPE inhibitory dose (IC 50 ) was computed in light of these findings 32 .We evaluated the cytotoxicity before this experiment by seeding cells in a 96-well culture plate at a density of 2 × 104 cells/well.The following day, the cells were exposed to culture media containing serially diluted samples.The medium was removed after 72 h, and the cells were washed with PBS.The subsequent procedures were performed identically to those previously described for the antiviral activity test.GraphPad PRISM software (GraphPad Software, San Diego, USA) was used to calculate the 50% inhibitory concentration (IC 50 ) and 50% cytotoxic concentration (CC 50 ) 33 .
Vol:.(1234567890) www.nature.com/scientificreports/Assessment of the antiviral activity against influenza virus (H1N1) Madin-Darby canine kidney (MDCK) cells and influenza virus (H1N1) were cultured in DMEM supplemented with 10% fetal bovine serum and 0.1% antibiotic/antimycotic solution.Fetal bovine serum, trypsin-EDTA, DMEM, and antibiotic and antimycotic solutions were obtained from Gibco BRL (Grand Island, NY, USA).The cytopathic effect (CPE), which was recently discovered, was utilized to assess antiviral activity and cytotoxicity assays utilizing the crystal violet method 34,35 .To summarize, one day before infection, MDCK cells were seeded at a density of 2 × 104 cells/well into a 96-well culture plate.The following day, the culture media was removed, and phosphate-buffered saline was used to wash the cells.The crystal violet method was used to monitor CPE, and the percentage of cell viability was calculated to estimate the infectivity of the H1N1 virus.Mammalian cells were treated with 0.1 mL of a diluted H1N1 virus suspension comprising CCID50 (1.0 × 10 6 ) of the virus stock.To achieve the necessary CPEs two days postinfection, this dosage was chosen.The cells were treated with extracts by adding 0.01 ml of media with the required extract concentration.The antiviral activity of each test sample was measured at doses that were diluted two times, beginning at 1000 μg/ml.Both the cell controls (noninfected, nondrug-treated cells) and the viral controls (virus-infected, nontreated cells) were included.The culture plates were cultured in 5% CO 2 at 37 °C for 72 h.The progression of cytopathic effects was observed via light microscopy.After being washed with PBS, the cell monolayers were fixed and stained with a 0.03% crystal violet solution in 2% ethanol and 10% formalin.Following cleaning and drying, the optical density of each well was measured using a spectrophotometer set at 570/630 nm.The following formula was used to determine the percentage of antiviral activity of the test compounds: antiviral activity × (mean optical density of cell controls − mean optical density of virus controls)/(optical density of test − mean optical density of virus controls) × 100 32 .The 50% CPE inhibitory dose (IC 50 ) was computed considering these findings.We evaluated the cytotoxicity before this experiment by seeding cells in a 96-well culture plate at a density of 2 × 10 4 cells/well.The following day, the cells were exposed to culture media containing serially diluted samples.The medium was removed after 72 h, and the cells were washed with PBS.The subsequent procedures were performed identically to those previously described for the antiviral activity test.GraphPad PRISM software (GraphPad Software, San Diego, USA) was used to calculate the 50% inhibitory concentration (IC 50 ) and 50% cytotoxic concentration (CC 50 ) 36 .

Assessment of the antiviral activity against the human herpes simplex virus type 2
Vero cells were cultured in DMEM supplemented with 0.1% antibiotic/antimycotic solution and 10% fetal bovine serum.Fetal bovine serum, trypsin-EDTA, DMEM, and antibiotic and antimycotic solutions were obtained from Gibco BRL (Grand Island, NY, USA).Using the previously discovered cytopathic (CPE) inhibitory effect, antiviral activity and cytotoxicity were assessed using the crystal violet method.To summarize, one day before infection, hep-2 cells were plated at a density of 2 × 10 5 cells/well in a 96-well culture plate.The following day, the culture media was removed, and phosphate-buffered saline was used to wash the cells.The crystal violet method, which measures CPE and allows for calculating the percentage of cell viability, was used to assess the infectivity of HSV-2.Mammalian cells were exposed to 0.1 mL of a diluted human adenovirus type 40 viral suspension comprising CCID50 (1.0 × 10 5 ) of the virus stock.Three days following infection, this dosage was chosen to result in the intended CPEs.The cells were treated with extracts by adding 0.01 ml of media with the required extract concentration.The antiviral activity of each test sample was assessed using a concentration range of 0.1-1000 μg/ ml that had been diluted twice.Both the cell controls (noninfected, nondrug-treated cells) and the viral controls (virus-infected, nontreated cells) were included.The culture plates were cultured in 5% CO 2 at 37 °C for 96 h.The progression of cytopathic effects was observed via light microscopy.After being washed with PBS, the cell monolayers were fixed and stained with a 0.03% crystal violet solution in 2% ethanol and 10% formalin.Following cleaning and drying, the optical density of each well was measured using a spectrophotometer set at 570/630 nm.Using the following formula, antiviral activity [(mean optical density of cell controls − mean optical density of virus controls)/(optical density of the test − mean optical density of virus controls)] × 100, the percentage of antiviral activities of the test compounds was determined following Pauwels et al. 32 .The 50% CPE inhibitory dose (IC 50 ) was computed in light of these findings.We evaluated the cytotoxicity before this experiment by seeding cells in a 96-well culture plate at a density of 2 × 104 cells/well.The following day, the cells were exposed to culture media containing serially diluted samples.The medium was removed after 72 h, and the cells were washed with PBS.The subsequent procedures were performed identically to those previously described for the antiviral activity test.GraphPad PRISM software (GraphPad Software, San Diego, USA) was used to calculate the 50% inhibitory concentration (IC 50 ) and 50% cytotoxic concentration (CC 50 ) 32 .

Molecular docking simulation
Antibacterial protein receptors were procured from the Protein Data Bank, as specified in Table 1, to examine the antibacterial properties of the promising compounds.Using PyMOL software, the crystal structures of the target receptors were preprocessed to exclude ions, water molecules, and preexisting ligands.The receptor molecule was then modified by adding hydrogen atoms using Autodock Vina 37 and stored in pdbqt format.Additionally, Open Babel was used to reduce each compound and convert it to a mol2 format 38 .The Autodock tools were then used to convert the compounds into pdbqt format.AutoGrid software was used to create ligand-centered maps with grid sizes of 90 Å × 90 Å × 90 Å.The remaining options were all set to their default values.AutoGrid and AutoDock Vina were used to construct the grid maps 37 .Additionally, Discovery Studio 4.5 software was used to investigate the 2-D bond interactions between the compounds and target receptors.Finally, using Lipinski's Rule of Five 39 , the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of the drugs were calculated.www.nature.com/scientificreports/Molecular dynamics (MD) simulation Protein-ligand complex binding affinities and interactions are frequently explained by molecular dynamics (MD) simulations along certain paths.In this study, GROMACS 2018 software was used to run 50 ns MD simulations to confirm the reliability and rationale of the docking results.The CHARMM36 force field parameters were utilized to design the topology of the viral protein 43 .The Geoff server was used to generate the Mangiferin coordinate files and topology.Following coordination, ligands are subject to limitations.At 300 K and 1.0 bar at atmospheric pressure, NVT and NPT equilibrium were measured for 500 and 1000 ps, respectively.Ultimately, each system underwent 50 ns MD simulations, with coordinate trajectories being recorded every 10 ps over the whole run.
The Root Mean Square Deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) were computed following the MD simulations.

Institutional review board statement
The methodology and experimental design were approved by the research ethics committee of Ahram Canadian University's College of Pharmacy, and the experiments were performed in compliance with their requirements.Affirmation Number: REC2024.

LC-MS/MS
LC-MS/MS was used to profile the phenolic metabolites in the floral aerial extracts of R. tuberosa and R. patula.Seventy-seven metabolites were detected in R. tuberosa and R. patula.combined.Based on their fragmentation pattern, molecular weight, and retention times as well as a comparison with published data from the literature and an online database called Mass Bank, the compounds were identified and provisionally annotated.According to the findings, phenolics and flavonoids are thought to be the two main active constituents found in both Ruellia species.R. patula has more flavonoids than R. tuberosa, with percentages of 57.8% and 38.7%, respectively, whereas R. tuberosa has higher proportions of phenolics than R. patula (49.3% and 36.4%,respectively).Compared with R. patula, R. tuberosa was shown to contain greater amounts of coumarins (8.5% and 1.2%), anthocyanidin glycosides (0.8% and 0.4%), and catechins (1.7% and 0.4%).Furthermore, R. patula was shown to contain higher levels of hydroxycinnamic acid and its glycosides (3.7% and 1.7%, respectively), as well as stilbenes and their glycosides (0.7% and 0.6%, respectively), than R. tuberosa.The results of the LC-MS/MS analyses are presented in Table 2 and Supplementary Fig. S1 and structures of the identified compounds are presented in Supplementary Figs.S2 and S3.

HPLC analyses
Seventeen and fourteen chemicals were found in the aerial extracts of R. tuberosa and R.

Antiviral activities of both Ruellia species
According to the results tabulated in Table 1 and Fig. www.nature.com/scientificreports/and oseltamivir as standard drugs for HAdV-40 75 , herpes simplex 76 , and H1N1 77 ., respectively.The results also showed a significant difference when compared with the values of standard drugs at P < 0.5.The results of the antiviral activities are tabulated in Table 4 and presented in Supplementary Figs.S5 and 6.

In silico pharmacokinetic ADME prediction of synthesized compounds
Based on the docking results, the most promising compounds, rutin, ellagic acid, catechin, hesperetin, and quercetin, with the highest affinity for ADME and toxicity risks were identified.First, the physiochemical properties of the tested compounds are shown in Table 5 and Fig. were examined and evaluated.Therefore, all the compounds possessed enough rotatable bonds (RBs 4), which is crucial for high structural flexibility.This is important because compounds with fewer than ten RBs are more likely to be bioavailable.As the number of RBs increases, they become more critical in determining successful interactions with certain binding sites.The hydrogen bond acceptors (HBAs) and donors (HBBDs) were also calculated for the three compounds, and it was found that all compounds had less than 10 HBA and less than 5 HBD, indicating a favorable balance of HBA and HBD and a greater likelihood of oral bioavailability.Additionally, the TPSA was evaluated as a metric for assessing drug transfer characteristics.The TPSA values of the compounds were found to be relatively high, with most falling in the optimal range of 60-140 for good absorption in the gut and oral bioavailability.Second, the lipophilicity and water solubility of the compounds rutin, ellagic acid, catechin, hesperetin, and quercetin were assessed.The obtained findings indicated that all the active compounds are highly soluble in water and have moderate solubility.The Log S values of rutin, ellagic acid, catechin, hesperetin, and quercetin ranged from − 4.666 to − 2.99, indicating high water solubility.The presence of soluble molecules simplifies the synthesis, handling, and formulation of bioactive substances.Additionally, www.nature.com/scientificreports/ the lipophilicity parameter LOGP of all the compounds appeared to fall within the allowed range of LOGP values between − 0.763 and 2.473.Third, tests were also conducted on the pharmacokinetics of the compounds.
The obtained results suggest that the examined compounds have high theoretical bioavailability and may be considered potential drug-like agents.However, all the compounds exhibited moderate intestinal absorption.
Additionally, they have the potential to interact with other drugs because they can suppress the enzymes CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4.Fourth, the study appears to have evaluated the drug-likeness of www.nature.com/scientificreports/compounds using various methods, including the Lipinski and Pfizer rules.Encouragingly, all the compounds met the drug-likeness requirements of Lipinski, indicating that they have desirable physicochemical properties for drug development and were rejected by Pfizer and the Golden Triangle rules.Regarding the distribution of compounds, many parameters, including plasma protein binding (PPB), were assessed.All compounds were present in more than 75% of the samples, with high protein-bound plasms having a low therapeutic index and a low fraction of unbound plasms (low: < 5%; middle: 5 ~ 20%; high: > 20%).Additionally, the blood-brain barrier (BBB) penetration of all compounds was calculated as the BBB, which cannot cross the blood-brain barrier.The volume distributions of all the compounds were obtained in the allowed range (0.04-20 L/kg).Finally, based on the computational assessment, it appears that the compounds are relatively safe and nontoxic (Table 6).

Molecular dynamics simulation (MDS)
Based on the docking of the three viral receptors with promising compounds, dynamic simulations were performed to investigate the behavior and stability of the protein complexes at the atomic level.First, several analyses of the MDS of a protease from human adenovirus (PDB: 4PIE) complexed with ellagic acid, hesperetin, and rutin were performed to assess the stability and dynamics of the 4PIE complexes.The RMSD was used to evaluate the stability of the protein structures.Figure 5A shows that 4PIE-Rutin was highly stable between 0.10 and 0.20 nm, while 4PIE-Hespertin and 4PIE-Ellagic acid exhibited stability between 0.10-0.25 nm and 0.15-0.25 nm, respectively.4PIE-Rutin was stabilized after 30 ns, 4PIE-Hespertin after 20 ns, and 4PIE-Ellagic acid after 15 ns.Additionally, RMSF analysis was used to assess the flexibility of amino acid residues during the simulation (Fig. 5B).
Most residues showed minimal fluctuations (0.1-0.3 nm), indicating relative stability.In addition, Rg analysis was performed to evaluate the overall shape of the protein complexes.Figure 5C shows Rg values ranging from 1.56 to 1.58 nm for 4PIE-Hespertin, 1.60-1.61nm for 4PIE-ellagic acid, and 1.65-1.67nm for 4PIE-rutin.The Rg values provide insights into the compactness or expansion of the protein structures during the simulation.Next, SASA analysis was conducted to understand the protein folding dynamics and stability.Figure 5D shows SASA values ranging from 85 to 95 nm for 4PIE-Hespertin, 88-95 nm for 4PIE-ellagic acid, and 105-115 nm for 4PIE-rutin.Furthermore, the intramolecular and intermolecular hydrogen bonds were analyzed to assess the stability of the complexes (Fig. 5E,F).The protease complexes with ellagic acid, hesperetin, and rutin initially formed a range of 140-170 intramolecular hydrogen bonds, which fluctuated during the simulation.In terms of intermolecular hydrogen bonds, ellagic acid formed the most interactions (2-14 bonds), followed by hesperetin (2-12 bonds) and rutin (2-8 bonds).These hydrogen bonds contribute significantly to the stability of the complex structures.Second, several MDS investigations were performed on Herpes simplex virus protease (1AT3) complexed with chlorogenic acid, hesperetin, and rutin to analyze the stability and dynamics of the complex.Figure 6A reveals that the RMSD values of 1AT3-chlorogenic were stable between 0.20 and 0.25 nm and stabilized after 10 ns, but those of 1AT3-Hespertin and 1AT3-rutin were stable between 0.20-0.30nm and 0.18-0.28nm and stabilized after 10 ns and 20 ns, respectively.Additionally, RMSF analysis was used to assess the flexibility of amino acid residues during the simulation (Fig. 6B).Most residues showed minimal fluctuations (0.1-0.3 nm), indicating relative stability.Figure 6C shows the Rg values for each complex, ranging from 1.60 to 1.63 nm for 4PIE-chlorogenic, 1.62 to 1.65 nm for 4PIE-Hesperetin, and 1.55 to 1.60 nm for 4PIE-rutin.Fourth, Fig. 6D shows the SASA values for each complex, ranging from 95 to 110 nm for 4PIE-chlorogenic and 4PIE-Hesperetin and from 93 to 105 nm for 4PIE-rutin.Figure 6E,F show that the number of intramolecular hydrogen bonds in the complexes with chlorogenic acid, hesperetin, and rutin initially ranged from 130 to 160 intramolecular hydrogen bonds, which fluctuated during the simulation.In terms of intermolecular hydrogen bonds, chlorogenic acid formed the most bonds (1-12 bonds), followed by hesperetin (2-10 bonds) and rutin (2-8 bonds).

Discussion
The potential impacts of phenolic compounds on a range of ailments, such as cancer, heart problems, stroke, and inflammation, and their antibacterial and antiviral properties have aroused a great deal of interest in medicinal plants 78 .Because it offers higher sensitivity and selectivity than other LC techniques, LC-MS/MS has been applied extensively in quantitative applications for numerous medicinal plants [79][80][81][82][83] .The richness of polyphenols, especially flavonoids and phenolic acids, was revealed by LC-MS/MS analyses of both Ruellia extracts.These analyses  www.nature.com/scientificreports/led to the identification of 77 phenolic chemicals, which were divided into the following categories: 53 flavonoids, 13 phenolic acids and their derivatives, 3 stilbenes, 4 coumarins, and 5 catechol compounds.Flavonols, Flavonoid-3-O-glucuronides, Flavonoid glucuronides, Flavonoid-O-p-coumaroyl glycosides, isoflavonoid glycosides, isoflavonoids, methylated flavonoids, and anthocyanidins were among the various kinds of flavonoids that were found.This result is in line with earlier studies that found flavonoids and phenolic acids to be two of the most important secondary metabolites in Ruellia species 25 .Both plant extracts contained luteolin, apigenin, quercetin, and p-coumaric acid, as previously described 25 .The semipolar character of both Ruellia extracts makes them suitable for the presence of polyhydroxy flavonoid aglycones and flavonoid glycosides with sugar units.), indicating the presence of hydroxyl and methyl groups from phenolic compounds and facilitating the identification of flavonoid subgroups 48,61 ..Phenolics are a group of secondary metabolites with different types of promising biological activities 85 .Phenolic acids are commonly reported metabolites in most profiling studies of medicinal plants.Phenolic acids generally produce the precursor ion [M − H] − , corresponding to deprotonated molecules, and the fragment ion [M-H-44] − , corresponding to the loss of CO 2 from the carboxylic acid group 86 .In this study, 13 phenolic acids were identified, including gallic acid, d-(−)-quinic acid, coumaric acid, 3,4-hydroxybenzoic     88 .To the best of the authors' knowledge, this is the first report on the detection of seventeen and fourteen phenolic compounds in the aerial extracts of R. tuberosa and R. patula, respectively, from flowering plants.
In this study, the floral aerial parts of R. tuberosa and R. patula were assessed for their potential for use as antiadenoviral agents for the treatment of adenovirus infections.Among the extracts tested, R. tuberosa showed the most potent anti-ADV activity (IC 50 of 20.65 µg/ml).Extracts of R. patula showed little activity against ADV (IC 50 equal to 32.26 µg/ml).Previous studies have also reported that the genus Ruellia has antibacterial and antifungal activities [21][22][23][24] .Phenolics and flavonoids such as gallic acid, ellagic acid, quercetin, and their conjugates, which were found in our work and other publications using various chromatographic techniques, may be responsible for the actions of both Ruellia species under investigation 25,89 .It has been demonstrated that certain naturally occurring quercetin molecules have antiviral or antibacterial properties.Research has also demonstrated that quercetin functions similarly against ADV-3 and ADV41.Quercetin has also been shown in earlier research to have antiviral effects on HIV, HSV, ADV3, ADV8, and ADV11.According to previous studies, the EC 50 values of pure substances for all anti-infective bioassays should be less than 25 mM 89 .
Numerous viruses, such as dengue virus, hepatitis B virus, herpes simplex virus, respiratory syncytial virus, parainfluenza virus, and adenovirus, were susceptible to the antiviral effects of flavonoids.It has been previously shown that these plant components interact with intracellular phases of the viral replication cycle in certain viruses.Regarding rotavirus, the glycone form of flavonoids works better than the aglycone form of these flavonoids.The flavonoid fraction had an inhibitory effect on the HSV-1 replication cycle and was effective against both HSV-1 and HSV-2.Our results may therefore suggest that the flavonoid concentrations of the aerial extracts of R. tuberosa and R. patula, which have promising anti-HSV-2 activity, are the cause of these findings 90 .
Phenolic compounds were used as sources of inspiration for the development of novel antiviral medications due to their antiviral efficacy against a variety of viruses.Recent studies have focused on the possible antiviral qualities of phenolic chemicals present in both Ruellia species.Numerous phenolic compounds found in Ruellia species have been demonstrated through independent studies to display antiviral action by inhibiting H1N1.R. tuberosa and R. patula showed anti-H1N1 viral activity at IC 50 s of 13.13 and 23.03 µg/ml, respectively, for both Ruellia species.Because they can interact with and inhibit the proteins and enzymes of both the virus and the host, phenolic substances have antiviral properties that prevent viral reproduction and infectivity 77 .Many studies have examined the antiviral properties of more than 100 selected phenolic compounds, among the numerous research groups studying the viral inhibitory effect of these compounds.These studies concluded that phenolic compounds with a high number of (-OH) groups and specific positions had the greatest inhibitory effect.Numerous substances possessing inhibitory properties against viruses have been identified in plants commonly used as traditional medicinal herbs or cuisines 91 .Chen et al. examined the bioavailability of phenolic compounds and discovered that, due to the high bacterial conjugative enzyme activity in the gastrointestinal tract environment, free phenolic compounds, such as quercetin, were assumed to partially degrade through dehydration reactions to hydroxybenzoic acids through gastric absorption 91 .Many of the phenolic compounds can be supplied through the colon, avoiding breakdown in the small intestine, and larger amounts can be found in the blood plasma by keeping the phenolic compounds bonded in a dietary matrix.Nevertheless, this adsorption technique provides only untargeted, low quantities of phenolic chemicals 87 .Gan et al. investigated the pharmacokinetic plasma concentration-time profiles for the adsorption of phenolic compounds from an extract of Echinacea purpurea in a rat model.They discovered that the ingestion of chlorogenic and caffeic acid causes adsorption to occur in 15 and 360 min, respectively, with corresponding disappearance half-lives of 7.72 and 6.00 h 91 .For more than thirty years, computer-aided drug design, or CADD, has gained popularity as a technique for designing, developing, and screening therapeutically significant molecular candidates 92 .These techniques, which aid in the more efficient optimization and screening of chemicals, combine several methodologies, such as molecular docking, toxicity, ADME (absorption, distribution, metabolism, and excretion), and molecular dynamics (MD) simulation.Currently, molecular docking is another well-liked technique that aids in determining how small molecule candidates interact with a target protein 91 .As a result, an in silico docking study was performed on the phenolic and flavonoid structures found in HPLC extracts of the aerial floral portions of R. tuberosa and R. patula against HADV-40, HSV-2, and H1N1 94 .The protease of HAdV-40 exhibited considerable binding affinity (− 7.20, − 6.90, and − 6.90 kcal/mol) when ellagic acid, rutin, and hesperetin were docked.Similarly, rutin, quercetin acid, and hesperetin showed affinities (− 8.90, − 8.60, and − 8.20 kcal/mol, respectively) for the H1N1 virus neuraminidase, whereas rutin, chlorogenic acid, and hesperetin showed affinities (− 7.90, − 7.00, and − 6.60 kcal/mol, respectively) for the HSV-2 protease.Additionally, the physicochemical characteristics and ADMET values of these potential compounds were computed.Additionally, MD simulations verified the stability of the complexes involving the neuraminidase of H1N1, proteases of HSV-2, and proteases of HAdV-40 (with RMSD of 0.10-0.25 nm, RMSF of 0.1-0.3nm, SASA of 85-115, and Rg of 1.56-1.67nm), proteases of HSV-2 (with RMSD of 0.18-0.30nm, RMSF of 0.1-0.3nm, SASA of 93-110, and Rg of 1.55-1.65 nm) and proteases of HAdV-40 (with RMSD of 0.10-0.25 nm, RMSF of 0.10-0.35nm, SASA of 135-155, and Rg of 1.90-1.98nm).

Conclusion
This study focused on the phytochemical profiling and antiviral activities of extracts from Ruellia species.Both in vitro and in silico approaches were used to explain the potential antiviral effects of these extracts.In the in vitro analysis, we examined the phytochemical composition of the Ruellia species extracts.They identified and quantified various bioactive compounds present in both extracts, which provided insights into their potential antiviral effects.Additionally, through computational docking and molecular dynamics simulations, valuable information
g, respectively) were detected only in the R. tuberosa extract, while pyrocatechol and quercetin (78.85 and 267.58 µg/g, respectively) were detected only in the R. patula extract.This approach also revealed that the concentrations of gallic acid, ellagic acid, naringenin, and cinnamic acid were greater in R. tuberosa (2878.71,2530.79,371.47, and 66.13 µg/g, respectively), while the concentrations of chlorogenic acid, methyl gallate, syringic acid, rutin, ferulic acid, and kaempferol were greater in R. patula

Table 3 .
Phenolic compounds in the aerial flowering parts of R. tuberosa and R. patula were identified by HPLC.RT retention time, RRT relative retention time to ellagic acid.a Area is expressed as the mean ± S.E.

Table 5 .
Prediction of the pharmacokinetics and physicochemical properties of the compounds.Oral bioavailability of the compounds determined with ADMETlab 2.0.Here, MW Molecular weight, nRig number of rigid bonds, fChar formal charge, nHet number of heteroatoms, MaxRing number of atoms in the largest ring, nRing number of rings, nRot number of rotatable bonds, TPSA topological polar surface area, nHA number of hydrogen bond acceptors, nHA number of hydrogen bond donors, logS Log of the aqueous solubility, logP Log of the octanol/water partition coefficient and logD logP at physiological pH 7.4.
Table1displays the number of chemicals observed in each ionization mode: five in the positive mode and 47 in the negative mode.This highlights how crucial it's to gather MS data across a broad spectrum of metabolites, ranging from basic to neutral and acidic, by employing both ionization techniques.More [M − H] − ions are produced by the flavonoid glycosides than [M + H] + ions.Their principal distinctive fragment ions resulting from the retroDiels Alder fragmentation pathway could be identified in their MS/MS spectra, along with losses of glycosyl moieties in both negative and positive ion modes.In terms of flavonoid glycosides, pentose (arabinose or xylose), rhamnose, hexose (glucose or galactose), and hexuronic or glucuronic acid were the common losses of 132, 146, 162, and 176 a.m.u., respectively.Additionally, flavonoids tended to be lost at 28 a.m.u.(CO), 18 a.m.u.(H 2 O), and 15 a.m.u.(CH 3

Table 6 .
Prediction of toxicity risks and oral toxicity prediction results of compounds.